The human erythrocyte membrane (RBCM) is the most thoroughly studied plasma membrane, not only because it serves as an accessible model of other human membranes, but also because inherited and acquired defects in its components lead to serious pathologies. Despite this scrutiny, fundamental aspects of the structure and function of the RBCM remain poorly understood. In Aim 1, we will characterize the structure and function of a newly discovered bridge (band 3 to 2-adducin to spectrin) that links the lipid bilayer to the spectrin-actin skeleton. We have recently demonstrated that ~1/3 of the band 3 population is anchored to the spectrin/actin junctional complex via this bridge and that rupture of the bridge leads to membrane fragmentation. To evaluate the significance of the bridge in vivo, we will map the binding site of adducin on band 3, identify mutations in band 3 that prevent adducin binding, generate a mouse containing the mutant band 3, and evaluate the morphological and mechanical properties of the mutant erythrocytes. Included as a major component of this aim is the characterization of a method to image the diffusion of single band 3 molecules in intact erythrocytes as a tool to sensitively monitor perturbations of RBCM structure. The membrane-spanning domain of band 3 (msdb3) not only mediates anion transport across the membrane, but also organizes a complex of membrane-spanning proteins, including glycophorin A, CD47, Rh proteins, aquaporin, and several transporters. In order to understand the function of msdb3 at a molecular level, we will solve its crystal structure at high resolution (This will be the first structure of any member of solute carrier class IV). We currently have crystals that diffract to <6E, and although we could solve a 6E structure now, we are confident that we can generate much higher resolution crystals using the novel strategies outlined in Aim 2. Strong evidence suggests that 1) red cell metabolism, 2) membrane structural properties, and 3) ion transport are regulated by O2. Because deoxyhemoglobin (but not oxyHb) binds with high affinity to band 3, and since band 3 associates with proteins responsible for each of the above properties, we hypothesize that the reversible association of band 3 with deoxyHb constitutes the "molecular switch" through which red cell oxygenation regulates membrane properties. In Aim 3, we will test this hypothesis using recently discovered band 3 mutations that either: i) eliminate all affinity for deoxyHb, or ii) enhance the affinity of deoxyHb for band 3 so significantly that deoxyHb can neither release its O2 nor dissociate from band 3, even at saturating O2. We propose to generate knock-in mice that express these two mutant band 3s, and use the mice to determine if the above O2- regulated properties are permanently "switched on" or permanently "switched off", as predicted. PUBLIC HEALTH RELEVANCE: The red blood cell performs a variety of functions critical to our survival, including transport of oxygen from the lungs to the tissues and carbon dioxide from the tissues to the lungs, delivery of nitric oxide to facilitate blood flow, participation in blood clotting to facilitate wound healing, and regulation of a number of other reactions that occur in the blood. Our research seeks to understand the molecular basis of each of these important functions, and where possible, to define clinical interventions that might enable treatment of conditions where the above processes malfunction.